自噬在激活芳香烃受体诱导的心肌肥大中的作用

Effect of autophagy in activation of aromatic hydrocarbon receptor-induced cardiac hypertrophy

目的探讨激活芳香烃受体(aryl hydrocarbon receptor,AhR)在心肌肥大中的作用,及自噬是否参与该过程。方法选用H9C2心肌细胞培养,实验分为对照组(Control组)、二甲基亚砜组(DMSO组)、2-(1’H-吲哚-3’-羰基)噻唑-4-羧酸甲酯组(ITE组)、sh AhR+ITE组(先转染sh AhR质粒24 h后加入ITE)、空载+ITE组、自噬抑制剂组(3-MA+ITE组)。蛋白免疫印迹法(Western blotting)检测AhR、微管相关蛋白3(microtubule-associated protein 1 light chain 3,LC3)和p62的表达水平;荧光实时定量验证(q PCR)检测β-肌球蛋白重链(β-myosin heavy chain,β-MHC)的表达。结果与对照组比较,ITE组和空载+ITE组细胞AhR表达水平和β-MHC的基因表达水平明显升高,差异均具有统计学意义(P<0.01);sh AhR+ITE组AhR表达水平明显低于对照组,差异具有统计学意义(P<0.01);与对照组比较,sh AhR+ITE组中β-MHC的基因表达水平差异无统计学意义(P>0.05);与对照组相比,DMSO组和sh AhR+ITE组的LC3-II/I比值和p62表达水平无明显变化,差异无统计学意义(P>0.05);与对照组相比,ITE组和空载+ITE组中,p62蛋白表达水平明显下降,差异均具有统计学意义(P<0.01),LC3-II/I比值升高,差异有统计学意义(P<0.01);3-MA+ITE组中p62蛋白表达水平,低于DMSO组(P<0.01),高于空载+ITE组(P<0.01);3-MA+ITE组中LC3-II/I比值,低于空载+ITE组(P<0.01),与DMSO组相比,差异无统计学意义(P>0.05);与对照组比较,ITE组与空载+ITE组中β-MHC的表达水平明显升高,差异具有统计学意义(P<0.01);DMSO组、sh AhR+ITE组和3-MA+ITE组中β-MHC的表达水平与对照组相比均没有发生变化,差异无统计学意义(P>0.05)。结论激活心肌细胞内AhR受体可能通过上调心肌细胞内自噬水平诱导心肌肥大。

Objective To investigate the effect of aromatic hydrocarbon receptor(AhR)activation in myocardial hypertrophy and the role of autophagy in this process.Methods H9C2 myocardial cells were cultured.The experiment was divided into the control group,DMSO group,ITE group,shAhR+ITE group,no-load+ITE group and 3-MA+ITE group.The expression levels of AhR,microtubule-associated protein1 light chain 3(LC3)and p62 were detected by Western blotting.The expression of beta-myosin heavy chain(beta-MHC)was measured by real-time fluorescence quantitative PCR(qPCR).Results Compared with the control group,the expression of AhR and beta-MHC was significantly higher in ITE group and noload+ITE group(P<0.01),and the AhR expression was significantly lower in sh AhR+ITE group(P<0.01).There was no significant difference in the expression of beta-MHC between sh AhR+ITE group and the control group(P>0.05).Compared with the control group,there was no significant difference in LC3-II/I ratio and p62 expression between DMSO group and sh AhR+ITE group(P>0.05).Compared with the control group,the expression of p62 protein was decreased significantly(P<0.01),and the LC3-II/I ratio was increased in ITE group and no-load+ITE group(P<0.01).The expression of p62 protein in 3-MA+ITE group was lower than that in DMSO group(P<0.01),but higher than that in no-load+ITE group(P<0.01).LC3-II/I ratio in 3-MA+ITE group was lower than that in no-load+ITE group(P<0.01),but there was no significant difference when compared with DMSO group(P>0.05).Compared with the control group,the expression of beta-MHC was increased significantly in ITE group and no-load+ITE group(P<0.01).The expression level of beta-MHC in DMSO group,shAhR+ITE group and 3-MA+ITE group was no significantly different when compared with the control group(P>0.05).Conclusion Activation of AhR receptor in cardiac myocytes may induce cardiac hypertrophy by up-regulation of autophagy in cardiac myocytes.